Ward W H, Cook P N, Mirrlees D J, Brittain D R, Preston J, Carey F, Tuffin D P, Howe R
ICI Pharmaceuticals, Macclesfield, Cheshire, U.K.
Adv Exp Med Biol. 1993;328:301-11. doi: 10.1007/978-1-4615-2904-0_32.
Aldose reductase (aldehyde reductase 2, ALR2) is often isolated as a mixture of two forms which are sensitive (ALR2S), or insensitive (ALR2I), to inhibitors. We show that ICI 215918 ((2-6-dimethylphenylsulphonyl)-nitromethane) follows either noncompetitive, or uncompetitive kinetics with respect to aldehyde for ALR2S, or the closely related enzyme, aldehyde reductase (aldehyde reductase 1, ALR1). Similar behaviour is exhibited by two other structural types of aldose reductase inhibitor (ARI), spirohydantoins and acetic acids, when either aldehyde, or NADPH is varied. For ALR2S, we have demonstrated kinetic competition between a sulphonylnitromethane, an acetic acid and a spirohydantoin. Thus, different ARIs probably have overlapping binding sites. Published studies imply that ALR2 follows an ordered mechanism where coenzyme binds first and induces a reversible conformation change (E.NADPH-->E*.NADPH). Reduction of aldehyde appears rate-limited by the step E*.NADP+-->E.NADP+. Spontaneous activation converts ALR2S into ALR2I and increases kcat. This must be associated with acceleration of the rate-determining step. We now propose the following hypothesis to explain characteristics of ARIs. (1) Inhibitors preferentially bind to the E* conformation. (2) The ARI binding site contains residues in common with that for aldehyde substrates. When aldehyde is varied, uncompetitive inhibition arises from association at the site for alcohol product in the E*.NADP+ complex which has little affinity for the substrate. Any competitive inhibition arises from use of the aldehyde site in the E*.NADPH complex. (3) Acceleration of the E*.NADP+-->E.NADP+ step upon activation of ALR2 reduces steady state levels of E* and so decreases sensitivity to ARIs.
醛糖还原酶（醛还原酶2，ALR2）常以对抑制剂敏感（ALR2S）或不敏感（ALR2I）的两种形式的混合物被分离出来。我们发现，ICI 215918（（2 - 6 - 二甲基苯基磺酰基）硝基甲烷）对于ALR2S或密切相关的酶醛还原酶（醛还原酶1，ALR1）而言，相对于醛遵循非竞争性或反竞争性动力学。当醛或烟酰胺腺嘌呤二核苷酸磷酸（NADPH）变化时，另外两种结构类型的醛糖还原酶抑制剂（ARI），即螺乙内酰脲类和乙酸类，也表现出类似行为。对于ALR2S，我们已经证明了磺酰基硝基甲烷、乙酸和螺乙内酰脲之间的动力学竞争。因此，不同的ARI可能具有重叠的结合位点。已发表的研究表明，ALR2遵循一种有序机制，即辅酶首先结合并诱导可逆的构象变化（E.NADPH→E*.NADPH）。醛的还原似乎在E*.NADP⁺→E.NADP⁺这一步受到速率限制。自发激活将ALR2S转化为ALR2I并增加催化常数（kcat）。这必然与限速步骤的加速相关。我们现在提出以下假设来解释ARI的特性。（1）抑制剂优先结合到E构象。（2）ARI结合位点包含与醛底物结合位点相同的残基。当醛变化时，反竞争性抑制源于在E.NADP⁺复合物中醇产物位点的结合，该复合物对底物几乎没有亲和力。任何竞争性抑制都源于在E*.NADPH复合物中醛位点的使用。（3）ALR2激活后E*.NADP⁺→E.NADP⁺步骤的加速降低了E*的稳态水平，因此降低了对ARI的敏感性。
